Immunity enhancing lactic acid bacteria

ABSTRACT

Novel bacteria Lactobacillus rhamnosus HN001 and HN 067, Lactobacillus acidophilus HN017, and Bifidobacterium lactis HN019 are claimed. Each strain provides immune enhancing effects when ingested.

This is the U.S. National Phase under 35 U.S.C. § 371 of InternationalApplication PCT/NZ98/00122, filed Aug. 18, 1998 which claims priority ofAustralian Application Nos. AU PP 3225, filed Apr. 28, 1998 and AU PO8699, filed Aug. 21, 1997.

TECHNICAL FIELD

This invention relates to novel strains of lactic acid bacteria andtheir use in enhancing immunity.

BACKGROUND ART

The consumption of products containing lactic acid bacteria (LAB) isassociated with a range of health benefits including enhancement ofimmunity. There are thousands of strains of lactic acid bacteria butonly some strains exhibit health-promoting properties. The ability ofthese bacteria to tolerate acids and bile salts, adhere to mucosalepithelial cells, and to survive passage through the gastrointestinaltract is considered an important criterion for selection ofhealth-promoting strains. Only a few strains of lactic acid bacteriawith proven health benefits have been identified to date.

Strains of LAB showing good adhesion to the cells of the mucosalepithelium of the small intestine thereby lending themselves totherapeutic applications are known from New Zealand Patent 248057. Themicro-organisms described in this patent enhance both natural inununity(phagocyte function) and acquired immunity (antibody responses andlymphocyte proliferation responses).

It is desirable to have other LAB bacteria that enhance a broad spectrumof immune responses including phagocyte function.

It is an object of this invention to go some way towards achieving thesedesiderata or at least to offer the public a useful choice of immuneenhancing lactic acid bacteria.

DISCLOSURE OF THE INVENTION

Accordingly, in one aspect the invention may be said broadly to consistof a biologically pure culture of Lactobacillus rhamnosus HN001, AGALdeposit number NM97/09514 dated Aug. 18, 1997.

In another aspect the invention may be said broadly to consist of abiologically pure culture of Lactobacillus rhamnosus HN067, AGAL depositnumber NM97/01925 dated Feb. 17, 1998.

In another aspect the invention may be said broadly to consist of acomposition of a biologically pure culture of any one of Lactobacillusacidophilus HN017, AGAL deposit number NM97/09515 dated Aug. 18, 1997,Lactobacillus rhamnosus HN001, Lactobacillus rhamnosus HN067 orBifidobacterium lactis HN019, AGAL deposit number NM97/09513 dated Aug.18, 1997 in an immunostimulating concentration, with a physiologicallyacceptable excipient or diluent.

In one embodiment said composition contains any two or more of saidstrains.

Preferably said physiologically acceptable excipient or diluent is afood.

Preferably said food is any one of cultured milk, yoghurt, cheese, milkdrink or milk powder.

Alternatively said composition is a pharmaceutical composition and saidexcipient or diluent is pharmacologically acceptable excipient ordiluent.

Immunity enhancing, physiologically acceptable, biologically purestrains of homologues or mutants of any one of the strains:

Lactobacillus acidophilus HN017,

Lactobacillus rhamnosus HN001,

Bifidobacterium lactis HN 019, or

Lactobacillus rhamnosus HN067.

In another embodiment the invention may be said broadly to consist of amethod of enhancing natural and acquired immunity which comprisesadministering to a mammal any one of the above biologically purecultures at an immunostimulating dosage rate.

In another embodiment substantially biologically pure cultures of two orthree of the above-defined strains are present.

Preferably said culture is administered in the form of a compositionwith a physiologically acceptable excipient or diluent.

Preferably said physiologically acceptable excipient or diluent is afood.

Preferably said food is cultured milk, yoghurt, cheese, milk drink ormilk powder.

This invention may also be said broadly to consist in the parts,elements and features referred to or indicated in the specification ofthe application, individually or collectively, and any or allcombinations of any two or more of said parts, elements or features, andwhere specific integers are mentioned herein which have knownequivalents in the art to which this invention relates, such knownequivalents are deemed to be incorporated herein as if individually setforth.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows the effect of supplementation of mice with productfermented with L. rhamnosus HN001 or unfermented product containing L.rhamnosus HN001 on phagocyte activity of peripheral blood leukocytes asdescribed in example 5. BALB/c mice were fed on milk based dietscontaining 10⁹ cfa (per day) L. rhamnosus HN001 in either fermented orunfermented product for 14 days. Phagocytic activity of peripheral bloodleukocytes was determined using flow cytometry and fluorosceinisothiocyanate-labelled Escherichia coli. Values are mean±standarderror. Significant differences (ANOVA, the SAS program) from thecontrol: **P<0.0001.

FIG. 2 shows the effect of supplementation of mice with live L.rhamnosus HN001 or heat killed L. rhamnosuis HN001 on phagocyticactivity of peripheral blood leukocytes as described in example 7.BALB/c mice were fed on milk based diets and orally administered 10⁹ cfu(per day) of either live or heat killed L. rhamnosus HN001 for 14 days.Phagocytic activity of peripheral blood leukocytes and peritonealmacrophages were determined using flow cytometry and fluorosceinisothiocyanate—labelled Escherichia coli. Values are mean±standarderror. Significant differences (ANOVA, the SAS program) from thecontrol, **P<0.0001.

FIG. 3 shows the effect of supplementation of mice with L. rhamnosusHN001 or B. lactis HN019 on bacteria translocation in mice challengedwith S. typhimurium as described in example 8. Unsupplemented and B.lactis HN019, or L. rhamnosus HN001 supplemented BALB/c mice were orallychallenged with S. typhimurium following continuous dailysupplementation. Six days after challenge mice were humanely killed andtheir livers and spleens were harvested for monitoring bacterialtranslocation. Tissue suspensions from the harvested organs were thencultured on MacConkey agar plates for 24-48 hr prior to enumeration.Values are mean±standard error. Significant differences (ANOVA, the SASprogram) from the control: *P<0.05.

FIG. 4 shows the effect of supplementation of mice with L. rhamnosusHN001 or B. lactis HN019 on the phagocytic activity of peripheral bloodleukocytes from mice challenged with S. typhimurium as described inexample 8. Unsupplemented and B. lactis HN019, or L. rhamnosus HN001supplemented BALB/c mice were orally challenged with S. typhimuriumfollowing continuous daily supplementation. Phagocytic activity ofperipheral blood leukocytes was determined six days after challengeusing flow cytometry and fluoroscein isothiocyanate-labelled Escherichiacoli. Values are mean±standard error. Values (mean±standard error) withdifferent superscripts are significantly different (ANOVA, the SASprogram): P<0.01.

FIG. 5 shows the effect of supplementation of mice with L. rhamnosusHN001 or B. lactis HN019 on the proliferative responses of spleenlymphocytes from mice challenged with S. typhimuritim as described inexample 8. Unsupplemented and B. lactis HN019, or L. rhamnosus HN001supplemented BALB/c mice were orally challenged with S. typhimuriumfollowing continuous daily supplementation. Six days after challenge theproliferative responses of spleen lymphocytes were measuredcolourimetrically following the incorporation of 5-bromo-2′-debxyuridinefor the final 16 hrs of the 96 hr incubation. Values (mean±standarderror) with different superscripts are significantly different (ANOVA,the SAS program): P<0.01).

MODES OF CARRYING OUT THE INVENTION

Freeze dried cultures of the four bacterial strains have been depositedat the Australian Government Analytical Laboratories (AGAL), The NewSouth Wales Regional Laboratory, I Suakin Street, Pymble, NSW 2073,Australia. Details of the deposits are:

Strain Number Date L. acidophilus HN017 NM97/09515 August 18, 1997 L.rhamnosus HN001 NM97/09514 August 18, 1997 B. lactis HN019 NM97/09513August 18, 1997 L. rhamnosus HN067 NM97/01925 February 11, 1998

The four strains identified above have been found to enhance a broadspectrum of immune responses including both natural and acquired immuneresponses.

EXAMPLE 1 Morphology and General Properties

RAPD analysis, 16S rRNA sequencing and SDS-PAGE analyses were used toconfirm taxonornical characterisation of strains. It was also found thatL. acidophilus HN017 was genetically different from L. acidophilus (LC1)of New Zealand Patent No. 248057.

RAPD analysis, 16S rRNA sequencing and SDS-PAGE analyses were used toconfirm taxonomical characterisation of L. rhamnosus HN067;species-specific primers used for characterisation of L. rhamnosus HN067at molecular level included Pr I (forward) 5-CAGACTGAAAGTCTGACGG-3 andPha II (reverse) 5-GCGATGCGAATTTCTATTATT-3.

The morphology and sugar fermentation properties of this strain aredetailed in Tables 1 and 2.

TABLE 1 Morphology and other characteristics L. acidophilus L. rhamnosusB. lactis L rhamnosus HN017 HN001 HN019 HN067 Short to medium Short tomedium Microaerophilic to Short to medium rods with rounded rods withsquare anaerobic rods with rods with square ends, generally ends inchains, characteristic shapes ends in chains, occurring singly generally0.7 × 1.1 × such as middle generally 0.7 × 1.1 × or in pairs or short2.0-4.0 μm, enlarged cells, ‘V’ or 2.0 to 4.0 μm, chains, when whengrown in palisade arrangement when grown in grown in MRS MRS broth. ofcells when grown MRS broth. broth. Gram positive, on TPY agar slabs.Gram positive, Gram positive, non-mobile, non- In MR5 broth withcatalase negative, non-spore spore forming, 0.05% cysteine non-mobile,non- forming, catalase catalase negative hydrochloride, theyspore-forming, negative facultative form middle-enlarged facultativefacultatively anaerobic rods cells and club shaped anaerobic rodsanaerobic rods with optimum (spatulated with optimum growth with optimumgrowth extremities) cells. temperature of growth temperature of Grampositive, non- 37 ± 1° C. and temperature of 37 ± 1° C. and motile andnon-spore optimum pH of 6.0 37 ± 1° C. and optimum pH of forming,catalase to 6.5. These are optimum pH of 6.0-6.5. These negative rodswith facultatively 6.0-6.5. These are facultatively optimum growthheterofermentative are obligately heterofermentative temperature ofbacteria and no homofermentative bacteria and no 37 ± 1° C. and optimumgas produced from bacteria and no gas produced from pH of 6.0-7.0.glucose. gas is produced glucose. Fructose-6-phosphate from glucose.phospho-ketolase positive.

TABLE 2 Carbohydrate fermentation pattern of selected Lactobacillus andBifidobacterium strains S1. No. Name of the bacterium Score* 1 L.acidophilus HN017 5755546 2 L. rhamnosus HN001 5757177 3 B. lactis HN0191051622 4 L. rhamnosus HN067 5757175 API 50 CH sugar fermentation kitwas used to determine the sugar-fermentation pattern. *The scores arebased on scores of 22 prominent sugars (Bergey's manual)

EXAMPLE 2 Adhesion to Intestinal Cells

The ability of probiotic strains to adhere to human intestinalepithelial cells (HT-29 and CaCo-2) was assessed in vitro usingdifferentiated cell-lines. Monolayers of HT-29 and CaCo-2 cells weregrown on cover slips and placed in multi-well dishes. 10⁸ cfu/ml of LABin 1 ml of spent culture supernatant was then added to cell layers alongwith 1 ml of DMEM medium and incubated for 1 hr at 37° C. in 10% CO₂-90%air. Monolayers were washed 4 times with PBS, fixed in methanol, Gramstrained and the number of bacteria adhering to epithelial cellsdetermined microscopically. On average, 20 fields were counted and theresults are summarised in Table 3.

TABLE 3 Adherence to HT-29 and CaCo-2 cell lines* STRAIN HT-29 CaCo-2 L.acidophilus HN017  98 ± 17 171 ± 16 L. rhamnosus HN001 161 ± 18 218 ± 35B. lactis HN019 188 ± 27 194 ± 25 *Number (mean ± SEM) of bacteria/100epithelial cells

EXAMPLE 3 Enhancement of Natural and Acquired Immunity

The immunoenhancing effects of the three strains L. rhamnosus HN001, L.acidophilus HN017 and B. lactis HN019 were examined by determiningphagocyte (blood leukocytes and peritoneal macrophage) function, andquantifying concentrations of specific antibodies to protein antigensused for mimicking responses to vaccines in mice.

The following experimental protocol was used:

1. Six-to-seven week old BALBIc mice, weighing 20-30 g were used.

2. Mice were randomly allocated to different treatment groups (Table 4)

3. Mice were fed L. acidophilus HN017, L. rhamnosus HN001 or B. lactisHN019 (10⁹ cfu/day) in 50 μl skim milk for 10 days. Control micereceived 50 μl of skim milk powder only.

4. All mice received skim milk powder based diet throughout theexperiment.

Blood leukocytes and macrophages from mice receiving L. acidophilusHN017, L. rhamnosus HN001 or B. lactis HN019 showed significantlygreater phagocytic capacity compared with cells from control mice (Table4). The production of oxygen radicals (oxidative burst) by leukocytesfrom probiotic fed mice was also higher than the control mice (data notshown).

TABLE 4 The effect of dietary L. acidophilus HN017, L. rhamnosus HN001and B. lactis HN019 on phagocyte function in mice % Blood % Peritonealleukocytes with macrophage with Treatment phagocytic activity phagocyteactivity Control 14.33 ± 0.87  66.1 ± 3.5  L. acidophihilus HN017  22.7± 1.21** 79.0 ± 1.0** L. rhamnosus HN001 24.84 ± 0.93**  0.5 ± 1.8** B.lactis HN019 23.19 ± 0.95** 77.4 ± 2.6* 

BALB/c mice were orally administered with 10⁹ cfu (per day) L.acidophilus HN017, L. rhamnosus HN001 or B. lactis HN019 for 10 days.Phagocytic activity of blood leukocytes and peritoneal macrophages wasdetermined using flow cytometry and fluorescein isothiocyanate—labelledEscherichia coli. Values are mean±standard error. Significantdifferences (Students t test) from the control: *P<0.05, **P<0.01.

The concentration of specific IgG antibodies in the sera and in theintestinal washings of mice receiving L. acidophilus HN017, L. rhamnosusHN001 or B. lactis HN019 was also greater than those of control mice(Table 5).

TABLE 5 The effect of dietary L. acidophilus HN017, L. rhamnosus HN001and B. lactis HN019 on serum and mucosal antibody responses SerumMucosal antibody response antibody response Treatment (units/ml)(units/ml) Control 80.2 ± 6.0   1350 ± 96.0  L. acidophilus 134.6 ±25.2*  1548 ± 270.0 HN017 L. rhamnosus HN001 118.5 ± 12.5**  1512 ±198.0 B. lactis HN019 158.1 ± 51.6*** 1548 ± 234.0

BALB/c mice were orally administered with 10⁹ cfu (per day) L.acidophilus HN017, L. rhamnosus HN001 or B. lactis HN019 for 10 days.Mice were immunised with cholera toxin (an antigen used to mimic entericinfection) on days 0 and 7. The concentration of specific antibodies inserum and intestinal secretions were measured using ELISA on day 10.Values represent mean±standard error. Significant differences (Studentst test) from control: *P=0.08; **P<0.05; ***P<0.01.

EXAMPLE 4 Immunostimulating Effects Following Supplementation with LABfor Four Weeks

The immunostimulating effects of L. acidophilus HN017, L. rhamnosusHN001 , and B. lactis HN019 were assessed in mice using the followingexperimental protocol:

1. Six-to-seven week old BALB/c mice, weighing 20-30 g were used.

2. Mice were randomly allocated (18/group) to different treatmentgroups.

3. After acclimatisation (for 7 days), mice were given 10⁹ cfu (per day)L. acidophilus HN017, L. rhamnosus HN001 , or B. lactis HN019, in 50 μlskim milk, for 28 days (from day 0 to day 28). Control mice received 50μl skim milk (without any micro-organisms) only.

4. Mice were offered a skim milk powder based-diet and water ad libitum,throughout the experiment.

5. Immunostimulating effects were assessed by monitoring phagocyticactivity of blood leukocytes and peritoneal macrophages, NK-cellactivity of splenic lymphocytes, lymphocyte proliferation (spleen cells)responses to a T-cell mitogen, ConA (an indicator of cell-mediatedimmunity) and antibody responses to Tetanus vaccine.

As seen in Table 6, leukocytes (neutrophils, monocytes and macrophages)from mice receiving L. acidophilus HN017, L. rhamnosus HN001, or B.lactis HN019 exhibited significantly greater phagocytic activity (anindicator of natural immunity) than leukocytes from control mice.

TABLE 6 The effect of dietary L. acidophilus HN017, L. rhamnosus HN001,and B. lactis HN019 in mice % Blood % Peritoneal leukocytes withmacrophages with Treatment phagocytic activity phagocytic activityControl 15.5  72.67  L. acidophilus HN017 29.4** 82.2*  L. rhamnosusHN001 24.2** 82.8**  B. lactis HN019 31.1** 83.0** 

Mice (18/group) were given 10⁹ cfu (per day) L. acidophilus HN017, L.rhamnosus HN001 , or B. lactis HN019 in 50 μl skim milk for 28 days.Phagocytic activity of blood leukocytes/peritoneal macrophages wasdetermined on day 28 using flow cytometry and fluoresceinisothiocyanate-labelled E. coli. Values are least square means.Significant differences (the SAS analysis): *P<0.002, **P<0.0005.

Consumption of L. acidophilus HN017, L. rhamnosus HN001, or B. lactisHN019 for 28 days also resulted in an increase in the NK-cell activity,lymphocyte proliferation responses to ConA and antibody responses toTetanus vaccine. For all these indicators of immunocompetence, micereceiving L. acidophilus NN017, L. rhamnosus HN001 , or B. lactis HN019had higher responses than those of control mice (Table 7).

Together these results show that supplementation for extended periodswith L. acidophilus HN017, L. rhamnosus HN001, or B. lactis HN019 isable to induce a sustained enhancement in several aspects of natural andacquired immunity.

TABLE 7 The effect of dietary L. acidophilus HN017, L. rhamnosus HN001,and B. lactis HN019 on NK cell activity and lymphocyte proliferationresponses to ConA and antibody responses to Tetanus vaccine. LymphocyteAntibody NK cell proliferation to responses to ConA activity ConATetanus vaccine Treatment (%) (absorbance) (units/ml) Control  8.8  1.4± 0.125 402.5 ± 41.4  L. acidophilus HN017  9.9 1.6 ± 0.44 923.9 ±116.0* L. rhamnosus HN001 11.5 1.8 ± 0.1* 711.5 ± 127.2* B. lactis HN01910.5 1.7 ± 0.5 844.6 ± 134.7*

Mice (18/group) were given 10⁹ cfu (per day) L. acidophilus HN017, L.rhamnosus HN001 , or B. lactis HN019 in 50 μl skim milk for 28 days(i.e. from days 0 to 28). NK-cell activity of splenic lymphocytes wasdetermined on day 28 using flow cytometry and D275-labelled Yac-1 cells.Lymphocyte proliferation responses of splenic lymphocytes to ConA wereassessed on day 28 using a commercial cell proliferation kit (BoehringerMannheim, Germany). For antibody responses, mice were immunised withTetanus vaccine (50 μl/dose, CSL, Australia) on days 7 and 21. Theconcentration of specific antibodies were determined using an ELISA;antigen supplied by the vaccine manufacturers (CSL, Australia) was usedfor coating plates. Values are least square means of 18 mice.Significant differences (the SAS analysis): *P <0.05.

EXAMPLE 5 Enhancement of Natural and Acquired Immunity Using FermentedVersus Unfermented Products

The aim was to assess the immunoenhancing efficacy of yoghurt made(fermented) using the probiotic strain L. rhamnosus HN001 compared tounfermented product containing L. rhamnosus HN001. The immunoenhancingeffects were examined by determining the phagocyte function (peripheralblood leukocytes and peritoneal macrophages) and lymphocyteproliferative responses to a B-cell mitogen (LPS).

The following experimental protocol was used:

1. Six-to-seven week old BALB/c mice, weighing 20-30 g were used.

2. Mice were randomly allocated to different treatment groups.

3. Control mice received a whole milk powder-based diet throughout theexperiment.

4. Test mice received 2.5 g yoghurt made using L. rhamnosus HN001 (10⁹cfu/day) or 2.5 g whole milk containing L. rhamnosus HN001 (10⁹ cfu/day)per day as well as a whole milk powder based diet for 14 days.

Results

Mice receiving yoghurt made with L. rhamnosus HN001 or whole milkcontaining L. rhamnosus HN001 displayed a significantly higher level ofphagocytic activity of peripheral blood leukocytes than was observed inmice receiving the control diet (FIG. 1). This increase was seenirrespective of whether the L. rhamnosus HN001 was delivered in theyoghurt (fermented with L. rhamnosus HN001) or unfermented productcontaining L. rhamnosus HN001. There was no difference in the level ofphagocytic activity between mice receiving the fermented yoghurt madeusing L. rhamnosus (HN001 ) compared to unfermented WMP productcontaining L. rhamnosus (HN001).

Both the unfermented and L. rhamnosus HN001 fermented product fed miceshowed higher lymphocyte proliferative responses to LPS than the controlmice (Table 8). There was no significant difference in the responsebetween mice receiving unfermented product containing L. rhamnosus HN001and mice receiving product fermented with L. rhamnosus HN001.

TABLE 8 The effect of fermented and unfermented L. rhamnosus HN001 onlymphocyte proliferative responses in mice Lymphocyte proliferation toLPS Treatment (absorbance) Control (WMP) 0.4699 ± 0.028 WMP Fermentedwith 0.5361 ± 0.028 L. rhamnosus HN001 Unfermented WMP with L. rhamnosus0.5518 ± 0.028* HN001

BALB/c mice were fed on milk based diets containing 10⁹ cfu (per day) L.rhamnosus HN001 in either unfermented product or yoghurt made with L.rhamnosus HN001 (fermented product) for 14 days. Control mice receivedmilk-based diet without any LAB. Proliferative responses were measuredcolourimetrically following the incorporation of 5-bromo-2′-deoxyuridinefor the final 16 hrs of the 96 hr incubation.

Values are means±standard error. Significant differences (Students ttest) from the control: *P=0.05.

Together these results suggest that supplementation with L. rhamnosusHN001 enhances a range of immune functions including phagocytic activityand lymphocyte cell proliferation. L. rhamnosus HN001 presented ineither fermented or unfermented product is effective at elicitingenhancement of immune function, with fermented product giving a greaterresponse for some functions and unfermented being superior in others.

EXAMPLE 6 Enhancement of Natural and Acquired Immunity by L. rhamnosusHN067

Experiment 1.

The immunoenhancing effects of L. rhamnosus HN067 were examined bymonitoring phagocytic capacity of peripheral blood leukocytes andperitoneal macrophages (indicator of non-specific immunity), andquantifying concentrations of specific antibodies to an immunisationantigen, cholera toxin (used for mimicking responses to entericvaccines) in mice.

The following experimental protocol was used:

1. Six-to-seven week old BALB/c mice, weighing 20-30 g were used. Theywere fed on a skim milk-based diet throughout the experiment.

2. Mice in the test group (n=6) were orally administered L. rhamnosusHN067 (10⁹ cfu/day) in 50 μl skim milk for 10 days. Control mice (n=6)received 50 μl of skim milk powder (without any LAB) only.

Results

Blood leukocytes and peritoneal macrophages from mice receiving L.rhamnosus HN067 showed significantly greater phagocytic activity(enhanced phagocyte function) compared with cells from control mice. Theresults are set out in Table 9 below.

TABLE 9 The effect of dietary L. rhamnosus HN067 on phagocyte function %Blood % Peritoneal leukocytes with macrophages with Treatment phagocylicactivity phagocytic activity Control 13.1 ± 1.5 76.4 ± 1.9 L. rhamnosusHN067 23.7 ± 1.5** 87.2 ± 1.9*

BALBIc mice (6/group) were fed on milk-based diet with or without oraladministration of L. rhamnosus HN067 (10⁹ cfu/day) for 10 days.Phagocytic activity of blood leukocytes and peritoneal macrophages weredetermined using flow cytometry and fluorescein isothiocyanate—labelledE. coli. Values represent least square mean±standard error LSM.Significant differences (the SAS program) from the control: *P=0.0005,**P=0.0001.

The concentration of specific antibodies to cholera toxin, an antigenused for oral immunisation, in the sera and in the intestinal washingsof mice receiving L. rhamnosus HN067 was also significantly greater thanthose of control mice (Table 10).

TABLE 10 The effect of dietary supplementation with L. rhamnosus HN067on serum and mucosal antibody responses to cholera toxin Mucosalantibody Serum antibody response response Treatment (units/ml)(units/ml) Control 63.1 ± 43.2  1969.7 ± 279.5  L. rhamnosus HN067 246.5± 43.2** 2995.5 ± 465.2*

BALB/c mice were fed on milk-based diet with or without L. rhamnosusHN067 (10⁹ cfu/day) for 10 days. Mice were immunised orally with choleratoxin (10 μg/dose), an antigen used to mimic enteric infection, on days0 and 7. Antibody levels in serum and intestinal secretions weremeasured using ELISA on day 10. Values represent least squaremean±standard error LSM. Significant differences (the SAS program) fromcontrol: *P=0.02; **P=0.0039.

Experiment 2.

The immunostimulating effects of L. rhamnosus HN067 were assessed inmice using the following experimental protocol:

1. Six-to-seven week old BALB/c mice, weighing 20-30 g were used. Theywere offered skim milk powder based diet and water ad libitum,throughout the experiment.

2. After acclimatisation for 7 days, mice in group 1 (n=20) were orallyadministered with 10⁹ cfu (per day) L. rhamnosus (HN067) in 50 μl skimmilk (group 1 n=20) for 14 days. Control mice (group 2, n=20) receivedskim milk without any microorganisms.

3. Immunostimulating effects were assessed by monitoring phagocyticactivity of blood leukocytes and peritoneal macrophages, and spleenlymphocyte proliferation responses to phytohaemagglutinin (PHA) andlipopolysaccharide (LPS) (T and B-cell mitogens respectively).

Results

Blood leukocytes and peritoneal macrophages from mice receiving L.rhamnosus HN067 exhibited significantly greater phagocytic activity (anindicator of natural immunity) than leukocytes and macrophages fromcontrol mice (Table 11).

TABLE 11 The effect of dietary L. rhamnosus HN067 on phagocyte functionin mice % Blood % Peritoneal Leukocytes with macrophages with Treatmentphagocytic activity phagocytic activity Control 13.7 ± 0.07 64.6 ± 2.1L. rhamnosus HN067 22.5 ± 0.07** 75.8 ± 1.7*

BALB/c mice were fed on milk-based diet with or without oraladministration of L. rhamnosus HN067 (10⁹ cfu/day) for 14 days.Phagocytic activity of blood leukocytes/peritoneal macrophages weredetermined on day 14 using flow cytometry and fluoresceinisothiocyanate-labelled E. coli. Values represent least squaremean±standard error LSM. Significant differences (the SAS program):*P=0.002, **P=0.0001.

Mice receiving L. rhamnosus HN067 for 14 days also displayed higherlymphocyte proliferation responses to PHA and LPS compared with controlmice (Table 12).

TABLE 12 The effect of L. rhamnosus HN067 supplementation on lymphocyteproliferation responses to PHA and LPS ConA Lymphocyte LymphocyteTreatment proliferation to PHA proliferation to LPS Control 1.18 ± 0.080.99 ± 0.07 L. rhamnosus HN067 1.37 ± 0.07* 1.24 ± 0.06**

BALB/c mice were fed on milk-based diet with or without oraladministration of L. rhamnosus HN067 (10⁹ cfu/day) for 14 days.Lymphocyte proliferation responses of spleen cells to PHA and LPS wereassessed on day 14 using a commercial cell proliferation kit (BoehringerMannheim, Germany). Values represent least square mean±standard errorLSM. Significant differences (the SAS program): *P<0.08, **P<0.01.

In summary, mice receiving L. rhamnosus HN067 displayed significantenhancement of a range of host immune responses including leukocytephagocytic function, antibody responses to oral immunisation, andlymphocyte proliferation responses to T and B-cell mitogens. Bloodleukocytes (neutrophils and monocytes) and macrophages are majoreffectors of natural immunity and play a major role in protectionagainst microbial infections. A correlation between in vitro lymphocyteproliferation responses to mitogens (T- and B-cell mitogens) andimmunocompetence of an individual is also well documented. Therefore,these results suggest that supplementation with L. rhamnosus HN067 isable to enhance several aspects of natural and acquired immunity.

EXAMPLE 7 Enhancement of Natural and Acquired Immunity Using Live andHeat Killed L. rhamnosus HN001

The aim of the present study was to investigate the immunoenhancingeffects of the probiotic strain L. rhamnosus HN001 when presented ineither the live or heat killed form. The effect on immune function wasassessed by determining phagocytic activity of peripheral bloodleukocytes. The effect of live and heat killed L. rhamnosus HN001 onhumoral immunity was investigated by immunising mice with cholera toxin,and measuring the concentrations of specific antibodies produced.

The following experimental protocol was used:

1. Six-to-seven week old BALB/c mice, weighing 20-30 g were used.

2. Mice were randomly allocated to different treatment groups.

3. Control mice received a skim milk powder based diet throughout theexperiment.

4. Test mice receive either 10⁹ cfu/day of live L. rhamnosus HN001 or10⁹ cfu/day heat killed L. rhamnosus HN001 per day as well as a skimmilk powder-based diet for 14 days.

5. Mice were orally immunised with cholera toxin on day 0 and day 7 offeeding.

Results

L. rhamnosus HN001 feeding significantly enhanced the level ofphagocytic activity of peripheral blood leukocytes compared to micereceiving the control diet (FIG. 2). This increase was seen irrespectiveof whether the L. rhamnosus HN001 was delivered in the live or heatkilled form. There was no difference in the level of phagocytic activitybetween the mice receiving live L. rhamnosus HN001 compared to heatkilled L. rhamnosus HN001.

Feeding of both live and dead L. rhamnosus HN001 induced an increase inboth serum and mucosal antibody responses compared to the control mice.However, the level of response was significantly greater in the mice fedthe live L. rhamnosus HN001 (Table 13).

TABLE 13 The effect of live and heat killed L. rhamnosus HN001 on serumand mucosal antibody responses to Cholera Toxin in mice Serum antibodyMucosal antibody Treatment response (units/ml) response (units/ml)Control 88.69 ± 18.52 708.6 ± 146.9 Live L. rhamnosus HN001 214.89 ±62.33*  2054.5 ± 285.8*** Heat Killed 174.89 ± 44.78  1533.6 ± 319.3  L.rhamnosus HN001

BALB/c mice were fed on milk-based diets and orally administered 10⁹ cfu(per day) L. rhamnosus HN001 in either live or heat killed form for 14days. Control mice received no LAB. Mice were orally immunised withCholera Toxin on days 0 and 7. Antibody responses (serum and intestinalsecretions) were measured using an ELISA on day 14. Values aremean±standard error. Significant differences (Students t test) from thecontrol: *P=0.05, ***P=0.0005.

These results suggest that both live and heat killed L. rhamnosus HN001are able to enhance aspects of natural and acquired immunity in mice.

EXAMPLE 8 Anti-infection properties of B. lactis HN019 and L. rhamnosusHN001

The aims of the current study were to:

1. Assess the protection efficacy of B. lactis HN019 and L. rhamnosusHN001 against the gastrointestinal pathogen Salmonella typhimutrum.

2. Determine the role of immunostimulation induced by B. lactis HN019and L. rhamnosus HN001 in protection against S. typhimurium infection inmice.

Anti-infection properties were assessed by measurement of bacterialtranslocation to the liver and spleen. The immunoenhancing effects wereexamined by determining the phagocyte function (peripheral bloodleukocytes and peritoneal macrophages) and lymphocyte proliferativeresponses to a T-cell mitogen (PHA).

The following experimental protocol was used:

1. Six-to-seven week old BALBIc mice, weighing 20-30 g were used.

2. Mice were randomly allocated to 4 difference treatment groups andwere individually housed.

3. Al mice received a skim milk powder based diet throughout theexperiment.

4. Test mice commenced daily feeding of B. lactis HN019 or L. rhamnosusHN001 (10⁹ cfu/day) 7 days prior to challenge, and continued for theduration of the trial.

5. Mice administered with B. lactis HN019 or L. rhamnosus HN001 and acontrol group (no LAB) were orally challenged with Salmonellatyphimurium (ATCC 1772) 8×10⁵ cfu/day for 5 days starting on day 7.

6. An uninfected control group did not receive S. typhimurium challenge.

7. On day 6 after challenge mice were used for the measurement ofbacterial translocation to the liver and spleen, and for immune functionassessment.

Results

Both the B. lactis HN019 and L. rhamnosus HN001 supplemented mice showedsignificantly lower levels of bacterial translocation into the liver andspleen than the S. typhimurium alone fed mice (FIG. 3).

Challenge infection resulted in a significant suppression of phagocytefunction (FIG. 4); the phagocytic activity of control mice challengedwith S. typhimurium was significantly lower than that of the uninfectedmice. However, infection with S. typhimurium had no effect on thephagocytic ability of peripheral blood leukocytes of mice supplementedwith B. lactis HN019 or L. rhamnosus HN001 . This was shown by similarlevels of phagocytic activity in mice supplemented with B. lactis HN019or L. rhamnosus HN001 and challenged with S. typhimurium and the normaluninfected control mice.

Both the B. lactis HN019 and L. rhamnosus HN001 supplemented mice showedhigher lymphocyte proliferative responses to PHA than the S. typhimuriumchallenged control (FIG. 5). There was no significant difference in theresponse between mice receiving B. lactis HN019 or L. rhamnosus HN001and the uninfected control mice.

Together these results suggest that supplementation with B. lactis HN019or L. rhamnosus HN001 is able to confer protection against entericpathogens such as Salmonella typhimurium. Enhanced resistance toinfection is accompanied by an increase in immune performance.

What is claimed is:
 1. A biologically pure culture of eitherLactobacillus (L.) rhamnosus HN001 AGAL deposit number NM07/09514 orLactobacillus (L.) rhamnosus HN067 AGAL deposit number NM97/01925.
 2. Acomposition comprising at least one of the strains selected from thegroup consisting of L. rhamnosus HN001 AGAL deposit number NM97/09514,L. rhamnosus HN067 AGAL deposit number NM97/01925, Bifidobacterium (B.)lactis HN019 AGAL deposit number NM97/09513 and L. acidophilus HN017AGAL deposit number NM97/09515, in an amount effective to stimulate animmune response, and a physiologically acceptable excipient or diluent.3. The composition as claimed in claim 2 comprising at least two or moreof said strains.
 4. The composition as claimed in claim 2 wherein saidphysiologically acceptable excipient or diluent is a food.
 5. Thecomposition as claimed in claim 4 wherein said food is selected from thegroup consisting of cultured milk, yoghurt, cheese, milk drink and milkpowder.
 6. The composition as claimed in claim 2 wherein saidphysiologically acceptable excipient or diluent is also apharmacologically acceptable excipient or diluent.
 7. A physiologicallyacceptable pure culture of a strain, homologue, or mutant of at leastone of the strains selected from the group consisting of: L. acidophilusHN017, L. rhamnosus HN001, B. lactis HN019, and L. acidophilus HN067,having all of the identifying characteristics of said strains.
 8. Amethod of enhancing natural and acquired immunity which comprisesadministering to a mammal in need thereof at least one a biologicallypure culture of a strain of claim 1 or claim 7 in an amount effective tostimulate an immune response.
 9. The method of claim 8 wherein two ormore of the strains are administered.
 10. A method of enhancing naturaland acquired immunity which comprises administering to a mammal in needthereof a therapeutically effective amount of the composition of claim2.
 11. The method of claim 8 wherein said physiologically acceptableexcipient or diluent is a food.
 12. The method of claim 11 wherein saidfood is selected from the group consisting of cultured milk, yoghurt,cheese, milk drink and milk powder.